Expression and regulation of sex determining genes in the mouse. Veronica Mercedes Narvaez Padilla A thesis submitted for the Degree of Doctor of Philosophy 1996 Department of Developmental Genetics Department of Biology National Institute for Medical Research, University College London The Ridgeway, Mill Hill, Gower Street, London, NW7 lAA London, WCIE GET ProQuest Number: 10016705 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10016705 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 In memory of Pepe and Arturo. ...Many times Fve been alone and many times Fve cried. Anyway you’ll never know the many ways Fve tried... (Lennon & M cC artney) Little darling, it’s being a long cold lonely winter. Little darling, it feels like years since it’s been here. Here comes the sun, here comes the sun and I say It’s alright! (G. Harrison) Contents Contents ...................................................................................................................................................4 List of figures and tables .....................................................................................................................6 Acknowledgements ..........................................................................................................................8 A b str a c t .................................................................................................................................................11 Chapter 1: Introduction ................................................................................................................... 12 1.1 Sex ........................................................................................................................................12 1.2 Sex Determination vs Differentiation ...................................................................... 12 1.3 Environmental sex determination ............................................................................12 1.4 Genetic Sex Determination ............................................................................................13 1.4.1 Drosophila ........................................................................................................ 14 1.4.2 C. elegans..................................................................................................18 1.4.3 Mammals ........................................................................................................21 1.4.4 More variations in genetic sex determination .......................................21 1.5 Organogenesis of the genital system ............................................................ 21 1.5.1 The urogenital system ..................................................................................21 1.5.2 The indifferent gonad .......................................................................25 1.5.3 Origin, migration and fate of the primordial germ cells ................... 26 1.5.4 Testis Differentiation ................................................................................... 26 1.5.5 Ovary differentiation ................................................................................... 27 1.6 Testis Determining Factor ...........................................................................................28 1.7 Genes involved in sex determination or differentiation .............................29 1.7.1 S ry......................................................................................................................29 1.7.2 Sox-9 ...................................................................................................................36 1.7.3 Dax-1................................................................................................................. 37 1.7.4 AMH .............................................................................................................40 1.7.5 S F -i .................................................................................................................... 41 1.7.6WT-J..............................................................................................................41 1.8 Aims of this thesis ...............................................................................................41 Chapter 2: Materials and Methods ...................................................................................................43 2.1 Commonly used solutions and enzymes ....................................................................43 2.2 Bacterial strains, plasmids ......................................................................................... 43 2.3 Purification of DNA fragments from agarose gels ...............................................44 2.4 Spectrophotometric quantitation of nucleic acids .......................................................44 2.5 Recombinant DNA techniques........................................................................44 2.5.1 Preparation of competent bacteria .............................................................44 2.5.2 Transformation .............................................................................................44 2.5.3 Blunt End Ligations .....................................................................................44 2.5.4 Cohesive End Ligations ................................................................................45 2.5.5 Small scale plasmid isolation (minipreps): ..........................................45 2.5.6 Large Scale Plasmid Purification (maxipreps) ................................... 45 2.5.7 Sequencing ...................................................................................................... 45 2.6 PCR techniques................................................................................................................46 2.6.1 Isolation of genomic DNA for PCR ............................................................46 2.6.2 Oligonucleotides used: ................................................................................47 2.6.3 Primer combinations and annealing temperatures ........................... 48 2.6.4 Oligonucleotide Purification .....................................................................49 2.6.5 PCR procedure ................................................................................................49 2.6.6 RNA Purification for RT-PCR ..................................................................49 2.6.7 Reverse Transcription of RNA ..................................................................50 2.7 Probe Labelling and Southern hybridization ..........................................................50 2.7.1 DNA labelling ................................................................................................50 2.7.2 Oligonucleotide labelling ........................................................................... 50 2.7.3 DNA Transfer and Hybridization ..........................................................50 2.8 Protein detection .............................................................................................................51 2.8.1 Sample preparation ..................................................................................... 51 2.8.2 Electroblotting ...............................................................................................51 2.8.3 Detection of the protein with antibodies .................................................. 51 2.9 Production of transgenic mice ....................................................................................51 2.9.1 Mouse Stocks ...................................................................................................51 2.9.2 Microinjections of fertilized mouse eggs ................................................ 52 2.9.3 X-Gal Staining ...............................................................................................52 2.9.4 Paraffin sections ........................................................................................... 53 2.10 RNAse Protection ..........................................................................................................53 2.10.1 Probe Synthesis.............................................................................................53 2.10.2 Hybridization ................................................................................................53 2.10.3 Digestion ........................................................................................................54 2.10.4 Probes ..............................................................................................................54 Chapter 3: Siy regulation ..................................................................................................................55 3.1 Introduction ............................................................................................................... 55 3.2: DNAse Hypersensitive Site Mapping ..........................................................55